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Fusobacterium necrophorum, a Gram-negative, anaerobic bacterium, is an opportunistic animal and human pathogen that causes a variety of infections termed necrobacillosis. There are 2 subspecies of F. necrophorum (subsp. necrophorum and subsp. funduliforme) that differ morphologically and biochemically and in virulence. Leukotoxin, a secreted protein, is considered to be the major virulence factor. In camelids, F. necrophorum causes a variety of infections, generally involving the lips, tongue, pharynx, interdigital spaces, foot pad, larynx, mandible, or maxillary bones. The objective of the current study was to characterize the presumptive Fusobacterium isolates from a variety of necrotic infections in llama (Lama glama) and alpaca (Vicugna pacos) and determine whether the strains possess leukotoxin activities. A total of 7 isolates from alpaca and 2 isolates from llama were characterized. Based on growth characteristics in broth culture, and biochemical and polymerase chain reaction analyses, all 9 isolates belonged to subsp. necrophorum and possessed the putative hemagglutinin gene. Western blot analysis with antileukotoxin antibodies raised in rabbit showed the presence of leukotoxin protein in the culture supernatant of all isolates. Furthermore, flow cytometry of the culture supernatants demonstrated cytotoxicity to bovine and alpaca polymorphonuclear leukocytes (PMNs). The extent of cytotoxicity to either alpaca or bovine PMNs differed among camelid strains. The cytotoxicity of many of the camelid strains was higher (P < 0.05) toward alpaca PMNs compared to bovine PMNs. Fusobacterium necrophorum isolates from llama and alpaca are similar to bovine isolates, and leukotoxin may be a major virulence factor.
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Journal of Veterinary Diagnostic Investigation
25(4) 502 –507
© 2013 The Author(s)
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DOI: 10.1177/1040638713491407
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Brief Research Reports
Fusobacterium necrophorum is a Gram-negative, anaerobic,
and rod-shaped bacterium that causes a variety of suppura-
tive and necrotic infections, generally called necrobacillosis,
in animals and human beings.
9
Two subspecies of F. nec-
rophorum are recognized.
12
The subsp. necrophorum is more
frequently encountered in animal infections,
6
while clinical
isolates from human infections have the characteristics of
subsp. funduliforme.
13
The subsp. necrophorum is more viru-
lent
6
because of the presence and/or increased production of
potent virulence factors, such as leukotoxin, lipopolysaccha-
ride, and hemagglutinin.
9
Of these factors, leukotoxin is
considered to be the major virulence determinant in the
pathogenesis of animal infections.
9
In wild and captive animals, necrobacillosis has been
reported to cause infections of the oral cavity in white-tailed
deer (Odocoileus virginianus), mule deer (Odocoileus
hemionus), pronghorn antelopes (Antilocapra americana),
and blue duikers (Philantomba monticola; syn. Cephalophus
monticola fusicolor).
2,3,11,16
In camelids, F. necrophorum
infections occur on the lips, tongue, pharynx, interdigital
spaces, foot pad, larynx, mandible, or maxillary bones.
5
In
alpaca (Vicugna pacos), the organism has been reported to
cause osteomyelitis of mandibles and many disseminated
necrotic lesions.
5
Occasionally, the infection may be aspi-
rated to the lung, causing severe necrotizing pneumonia,
while infections in the interdigital space and foot pad can
lead to lameness.
5
The current study examined presumptive
Fusobacterium isolates from a variety of necrotic infections
in llama (Lama glama) and alpaca to identify the species and
subspecies, and to determine whether the strains possess leu-
kotoxin activity.
A total of 9 isolates, 2 from llama (61557 and 69521) and
7 from alpaca (11034, 61715, 68690, 68699, 73556, 118610,
and 1012060) were included in the study (Table 1). Fusobac-
terium necrophorum subsp. necrophorum strain A25 and
subsp. funduliforme, strain B35, both previously isolated
from liver abscesses of cattle,
6
served as reference cultures in
all the tests conducted to characterize the llama and alpaca
isolates. All strains were cultured in brain-heart infusion broth
a
(BHI) that was prereduced with 0.05% cysteine hydrochlo-
ride and anaerobically sterilized (PRAS).
15
The growth char-
acteristic (sedimentation or no sedimentation) of the isolates
491407JVD
XXX10.1177/1040638713491407Fusobacterium necrophorum from llama and alpacaKumar et al.
research-article2013
From the Departments of Diagnostic Medicine/Pathobiology (Kumar,
Amachawadi, Nagaraja, Narayanan) and Clinical Sciences (Anderson),
College of Veterinary Medicine, Kansas State University, Manhattan, KS.
1
Corresponding Author: Sanjeev Narayanan, K-246, Mosier Hall,
Department of Diagnostic Medicine and Pathobiology, Kansas State
University, 1800 Denison Avenue, Manhattan, KS 66506.
sanjeev@vet.k-state.edu.
Characterization of Fusobacterium
necrophorum isolated from llama and alpaca
Amit Kumar, David Anderson, Raghavendra G. Amachawadi,
Tiruvoor G. Nagaraja, Sanjeev K. Narayanan
1
Abstract. Fusobacterium necrophorum, a Gram-negative, anaerobic bacterium, is an opportunistic animal and human
pathogen that causes a variety of infections termed necrobacillosis. There are 2 subspecies of F. necrophorum (subsp.
necrophorum and subsp. funduliforme) that differ morphologically and biochemically and in virulence. Leukotoxin, a secreted
protein, is considered to be the major virulence factor. In camelids, F. necrophorum causes a variety of infections, generally
involving the lips, tongue, pharynx, interdigital spaces, foot pad, larynx, mandible, or maxillary bones. The objective of the
current study was to characterize the presumptive Fusobacterium isolates from a variety of necrotic infections in llama (Lama
glama) and alpaca (Vicugna pacos) and determine whether the strains possess leukotoxin activities. A total of 7 isolates from
alpaca and 2 isolates from llama were characterized. Based on growth characteristics in broth culture, and biochemical and
polymerase chain reaction analyses, all 9 isolates belonged to subsp. necrophorum and possessed the putative hemagglutinin
gene. Western blot analysis with antileukotoxin antibodies raised in rabbit showed the presence of leukotoxin protein in
the culture supernatant of all isolates. Furthermore, flow cytometry of the culture supernatants demonstrated cytotoxicity to
bovine and alpaca polymorphonuclear leukocytes (PMNs). The extent of cytotoxicity to either alpaca or bovine PMNs differed
among camelid strains. The cytotoxicity of many of the camelid strains was higher (P < 0.05) toward alpaca PMNs compared
to bovine PMNs. Fusobacterium necrophorum isolates from llama and alpaca are similar to bovine isolates, and leukotoxin
may be a major virulence factor.
Key words: Alpacas; Fusobacterium necrophorum subsp. necrophorum; leukotoxin; llamas.
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Fusobacterium necrophorum from llama and alpaca
503
in the PRAS-BHI broth was recorded, and the isolates were
tested biochemically using a commercial kit.
b
All isolates showed uniform turbidity in the broth cultures
(no sedimentation) and, based on the commercial identifica-
tion kit, all isolates were positive for indole production and
alkaline phosphatase activity, similar to subsp. necrophorum,
strain A25. Many of the isolates, except oral swab (73556)
and tongue abscess (1012060) isolates, obtained from llama
and alpaca were associated with infections involving the
bone. The alkaline phosphatase activity may help the organ-
ism in bone resorption creating a deep wound, and thus mak-
ing a more anaerobic environment for the bacterial growth.
17
The frequent association of F. necrophorum in camelids with
necrotic infections in bone needs further study and explanation.
To identify the organisms at the molecular level, different
polymerase chain reaction (PCR) assays were carried out
using a PCR machine.
c
The genus-specific PCR assay based
on the 16S ribosomal RNA gene
8
yielded an amplicon of 610
bp, confirming that all isolates belonged to genus Fusobacte-
rium (Fig. 1A). The subspeciation of the isolates was based
on PCR amplifications of hemagglutinin (haem) gene
1
and
the leukotoxin (lkt) operon promoter–containing intergenic
region.
18
The primer sequences, PCR running conditions,
and amplicon sizes are shown in Table 2. The PCR specific
for the hemagglutinin gene amplified a 315-bp product from
all llama and alpaca isolates and in the bovine strain of subsp.
necrophorum, but not in the bovine strain of subsp. funduli-
forme (Fig. 1B). The PCR assay designed to amplify the lkt
promoter–containing intergenic region of the subsp. nec-
rophorum amplified a product of 571 bp with all the isolates
and that of the bovine strain of subsp. necrophorum (Fig. 1C).
In contrast, the PCR assay designed to amplify the lkt
promoter–containing intergenic region of subsp. funduli-
forme gave a negative result with all of the camelid isolates
(Fig. 1D) and, as expected, amplified a product of 337 bp
with the bovine strain of subsp. funduliforme. A summary of
the PCR results is given in Table 1. Overall, the results
revealed that the Fusobacterium isolates from alpaca and
llama belonged to subsp. necrophorum. This is similar to a
previous observation that necrotic infections caused by
F. necrophorum in cattle are more often caused by subsp.
necrophorum.
9
The production of leukotoxin was tested by a Western blot
assay for the leukotoxin protein in culture supernatant. An
aliquot of each supernatant, containing 20 µg of protein as
determined by Bradford assay,
d
was loaded into each lane.
Rabbit polyclonal antiserum, raised against affinity-purified
leukotoxin from a bovine strain of subsp. necrophorum, was
used as the primary antibody
10
followed by incubation with
goat antirabbit immunoglobulin G conjugated with alkaline
phosphatase
e
as the secondary antibody. The culture superna-
tant was prepared by growing the isolates in PRAS-BHI
broth to an absorbance of 0.60–0.65 at 600 nm, pelleting
cells by centrifugation, filtering through a 0.22-µm filter,
f
and concentrating 60-fold with a 100-kDa molecular mass
cutoff filter.
f
The Western blot with concentrated superna-
tants from all strains showed 110 and 90 kDa bands, similar
to that of bovine strain of subsp. necrophorum A25 (Fig. 2).
A 250-kDa band was observed in strains 1012060, 61715,
69521, and 73556, but the band was absent in 11034, 68690,
68699, and 118610 strains and was faint in the bovine strain
of A25. The 150-kDa band was present in all strains except
in strains 11034, 68690, 68699, and 118610. Bands of 130
and 125 kDa were present in all strains, including the bovine
Table 1. Source of Fusobacterium necrophorum isolates from llama and alpaca and polymerase chain reaction (PCR) analysis results.
Leukotoxin gene promoter region PCR
specific for:
Animal/source Strain
Fusobacterium-
specific PCR
Hemagglutinin gene
(haem) PCR subsp. necrophorum subsp. funduliforme
Bovine
Liver abscess A25 + + +
B35 + +
Llama
Sequestrum of hind limb 61557 + + +
69521 + + +
Alpaca
Necrotic mandible 11034 + + +
Necrotic infection in maxillary bone 61715 + + +
of forelimb 68690 + + +
Tooth abscess, osteomyelitis of
mandible
68699 + + +
Oral swab 73556 + + +
Bone sequestrum 118610 + + +
Tongue abscess 1012060 + + +
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Kumar et al.
504
Figure 1. Polymerase chain reaction amplification of genes targeting Fusobacterium genus–specific 16S ribosomal DNA (A),
hemagglutinin (haem) gene (B), and leukotoxin (lkt) gene promoter specific for subsp. necrophorum (C) and for subsp. funduliforme
(D). Lane M: 100-bp DNA ladder; each lane (lanes 1–11) represents DNA template of different isolates. Lanes 1 (69521) and 2 (61557):
llama strains; lanes 3–9 (68699, 61715, 68690, 11034, 73556, 1012060, and 118610): alpaca strains; lanes 10 (subsp. necrophorum strain
A25) and 11 (subsp. funduliforme strain B35): bovine strains.
Table 2. Primers, target genes, polymerase chain reaction (PCR) running conditions, and amplicon sizes.
Primer Target PCR conditions
Product size
(base pairs) Reference
Fuso 1 (for): 5’-GAGAGAGCTTTGCGTCC-3’
Fuso2 (rev): 5’-TGGGCGCTGAGGTTCGAC-3’
Genus-specific 16S
ribosomal DNA for
Fusobacterium
94°C for 5 min; 35
cycles of 94°C for 30
sec, 60°C for 30 sec,
and 72°C for 30 sec;
and final extension at
72°C for 7 min
610 8
Haem (for):
5’-CATTGGGTTGGATAACGACTCCTAC-3’
Haem (rev):
5’-CAATTCTTTGTCTAAGATGGAAGCGG-3’
Putative hemagglutinin
gene specific for subsp.
necrophorum
95°C for 5 min; 35
cycles of 95°C for 30
sec, 65°C for 30 sec,
and 72°C for 30 sec;
and final extension at
72°C for 4 min
311 1
Fund (for):
5’-CTCAATTTTTGTTGGAAGCGAG-3’
Fund (rev):
5’-CATTATCAAAATAACATATTTCTCAC-3’
Promoter region of
leukotoxin gene
specific for subsp.
funduliforme
94°C for 3 min; 35
cycles of 94°C for
1 min, 52.2°C for
30 sec, and 64.4°C
for 30 sec; 72°C
for 1 min; and final
extension at 72°C for
4 min
337 18
5’lktpxmxh (for):
5’-GAAATCTTTAAAGCAC-3’
3’lktpxmxh (rev):
5’-CATAATTTCTCCCAATTTTATT-3’
Promoter region of
leukotoxin gene in
subsp. necrophorum
and subsp. funduliforme
94°C for 3 min; 35
cycles of 94°C for
1 min, 52.2°C for
30 sec, and 64.4°C
for 30 sec; 72°C
for 1 min; and final
extension at 72°C for
4 min
571 in subsp.
necrophorum
and 449
in subsp.
funduliforme
18
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Fusobacterium necrophorum from llama and alpaca
505
strain. In 2 strains, 68690 and 68699, all bands were gener-
ally of low intensity. One strain (1012060) showed additional
high-intensity bands of 80, 60, and 40 kDa. Alpaca strains
61715 and 73556 showed 2 additional bands in the 30-kDa
range, and the 30-kDa band was more prominent. These 2
bands were absent in all other strains (Fig. 2). The higher
intensity of protein bands from strain 1012060 could be
either due to difference in the promotor sequence leading to
a higher expression of leukotoxin or to difference in amino
acid sequence of the protein increasing its affinity to the anti-
body. However, the higher expression of leukotoxin by the
strain 1012060 was not expected because the cytotoxicity of
this strain on polymorphonuclear leukocytes (PMNs) was
less compared to 2 other camelid strains (as described
below). In addition, the multiple bands that appeared in the
current analysis supports the previous observation that the
leukotoxin from F. necrophorum is highly unstable.
13,15
The
difference in the banding pattern among the strains is most
likely because of cleavage by proteolytic enzymes.
14
Like-
wise, the difference in intensities of similar bands in different
strains could also be attributed to the difference in the proteo-
lytic activity.
The leukotoxicity of the culture supernatant was assessed
by a cell viability assay using propidium iodide in a flow
cytometer.
10
For the assay, PMNs collected from the periph-
eral blood of cattle and alpaca were used.
14
The viable PMN
(1 × 10
6
) cells were treated with culture supernatants for 45
min at 37°C and 5% CO
2
, washed twice with phosphate buff-
ered saline (PBS), resuspended in PBS, and stained with 10
ml of propidium iodide (50 mg/ml) in the dark for 5 min. The
PMNs in complete Roswell Park Memorial Institute (RPMI-
1640
g
) medium and PMNs treated with supernatants auto-
claved (to denature the protein) from each strain served as
negative controls. The samples were processed on a flow
cytometer,
h
and data were analyzed using commercial
software.
i
The flow cytometry analysis showed that the
culture supernatants from llama and alpaca isolates were
cytotoxic to alpaca and bovine PMNs (Fig. 3). The statistical
analysis of cytotoxicity was performed using a generalized
linear model (PROC GENMOD).
j
For analyzing the cytotox-
icity of the culture supernatants of the isolates to alpaca and
bovine PMNs, a repeated measures analysis of variance
model with a serial correlation structure was used. The val-
ues were assumed to be normal, and a generalized linear
model was used to fit the model. Score statistics based on the
differences in the least square means were used to assess the
significance of strains, species, and their interactions. Results
were considered significant at P < 0.05.
The extent of cytotoxicity with either alpaca or bovine
PMNs differed among camelid strains. The cytotoxicity of
many of the camelid strains were higher (P < 0.05) with
alpaca PMNs compared to bovine PMNs. Similarly, strain
A25 (subsp. necrophorum) was more cytotoxic to bovine
PMNs compared to alpaca PMNs. However, the cytotoxicity
of the B35 strain (subsp. funduliforme) did not differ between
alpaca and bovine PMNs. Also, the 2 bovine strains did not
differ significantly in cytotoxicity with alpaca PMNs, but
strain A25 was more cytotoxic than strain B35 with bovine
PMNs. With alpaca PMNs, the cytotoxicities of 7 out of 9
camelid strains were higher than either of the 2 bovine
strains, cytotoxicity of 1 strain (118610) was similar to, and
that of another strain (69521) was lower than that of bovine
strains (Fig. 3). With bovine PMNs, the cytotoxicities of all
camelid strains, except strain 73556, were lower than that of
the bovine strain, A25 (Fig. 3). There was no difference in
the cell viabilities of PMNs treated with the autoclaved cul-
ture supernatant of either camelid or bovine strains with
PMNs suspended in RPMI-1640 as negative control (data
not shown), suggesting the toxin is a protein and not lipo-
polysaccharide.
The cell viability assay using alpaca and bovine PMNs
showed that the culture supernatants of camelid strains were
Figure 2. Western blot analysis of culture supernatants of Fusobacterium necrophorum strains from llama and alpaca. Lane M: protein
ladder; lane 1: bovine strain, A25 (positive control); lane 2: 11034; lane 3: 1012060; lane 4: 61557; lane 5: 61715; lane 6: 69521; lane 7:
68690; lane 8: 68699; lane 9: 73556; lane 10: 118610.
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Kumar et al.
506
cytotoxic to PMNs of both alpaca and bovine. However,
many of the strains of llama and alpaca isolates were more
toxic to the alpaca PMNs compared to bovine PMNs, which
may indicate some animal species specificity with regard to
cytotoxicity. The species specificity of bovine F. necropho-
rum leukotoxin has been previously reported.
4,15
The impor-
tance of leukotoxin as a virulence factor in fusobacterial
infections in camelids needs to be assessed. There is a sug-
gestion that leukotoxin is not a universal virulence factor of
those strains of F. necrophorum responsible for necrotic
infections in animals and human beings.
7
In the current study,
all the isolates from llama and alpaca were shown to have a
functional leukotoxin. It is possible that, in camelids, leuko-
toxin plays a major role in the establishment of necrotic
infections. In conclusion, the present study has characterized
Fusobacterium isolates of camelid origin. The Fusobacte-
rium isolates from the necrotic infections in alpaca and llama
belong to the subsp. necrophorum and the strains had leuko-
toxin activity, which could be a major virulence factor.
Acknowledgements
Mr. Sailesh Menon is thanked for his help and suggestions with
manuscript preparation and submission. Dr. Greg Peterson is also
thanked for laboratory support.
Sources and manufacturers
a. BD, Franklin Lakes, NJ.
b. RapID ANA II kit, Remel Inc., Lenexa, KS.
c. Eppendorf Mastercycler Gradient, Fisher Scientific, Pittsburgh, PA.
d. Bio-Rad Laboratories, Hercules, CA.
e. Sigma-Aldrich, St. Louis, MO.
f. Millipore Corp., Billerica, MA.
g. Life Technologies, Grand Island, NY.
h. FACSCalibur flow cytometer, BD Bioscience, San Jose, CA.
i. Cell Quest Analysis Software, BD, Franklin Lakes, NJ.
j. SAS v. 9.3, SAS Institute Inc., Cary, NC.
Declaration of conflicting interests
The author(s) declare no conflicts of interest with respect to the
research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: The
SUCCESS-FYI grant from the College of Veterinary Medicine at
Kansas State University partially supported the study.
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Figure 3. Cytotoxicity assay with culture supernatants of bovine and camelid strains of Fusobacterium necrophorum subsp.
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different (P < 0.05) from both bovine strains with alpaca PMNs (A) and from bovine strain A25 with bovine PMNs (B).
a
Cytotoxicity
with bovine PMNs significantly different (P < 0.05) from alpaca PMNs.
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Fusobacterium necrophorum from llama and alpaca
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... F. necrophorum is considered a normal inhabitant of the alimentary tract of alpacas. 8,11 Two subspecies of this microorganism have been identified: F. necrophorum ssp. necrophorum (biotype A) and F. necrophorum ssp. ...
... 12,13 In a study performed with F. necrophorum isolates obtained from different necrotic lesions in alpacas and llamas, leukotoxin was demonstrated in the culture supernatant in all isolates, suggesting that this toxin may play a role in the pathogenesis of necrotic lesions in these animal species. 11 In ruminants, most of the ulcerative lesions in the upper digestive system that are associated with F. necrophorum occur after physical damage to tissues (e.g., trauma, ischemia, and parasitic or viral infections), which provides an anaerobic environment. 12,23,25 Once established in the tissue, F. necrophorum proliferates, causing extensive foci of coagulative necrosis. ...
... T. pyogenes and F. necrophorum are normally present on mucous membranes of the alimentary tract of camelids. 8,11 Pseudomonas spp. have been implicated in opportunistic infections and linked to environmental sources, particularly aqueous systems. ...
Article
Ulcers of the oral cavity, esophagus, and gastric compartments of South American camelids are uncommon. Multifocal-to-coalescing ulcers were identified in the oral cavity, esophagus, and/or gastric compartments of 5 alpacas submitted for postmortem examination. Fusobacterium necrophorum was isolated from the lesions in all alpacas, in combination with other aerobic and anaerobic bacteria. In 4 of these cases, F. necrophorum–associated lesions were considered secondary to neoplasia or other chronic debilitating conditions; in 1 case, the alimentary ulcers were considered the most significant autopsy finding. It is not known if this agent acted as a primary or opportunistic agent in mucosal membranes previously damaged by a traumatic event, chemical insult, immunodeficiency, or any other debilitating condition of the host.
... funduliforme was isolated from clinical human infections and their virulence is determined by secreting leukotoxin. In humans, F. necrophorum is responsible for Lemierre syndrome, which begins as bacterial pharyngitis and rapidly progresses to septic thrombophlebitis of the jugular vein [87][88][89][90]. Fusobacterium nucleatum was enriched in colorectal carcinoma. ...
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... Laryngeal lesions, comparable to those described in sheep, have been reported in calves [15], camelids [16], a white-tailed deer (Odocoileus virginianus) [17], thoroughbreds [18] and humans [19]. In calves, similar lesions are part of oral necrobacillosis, which is caused by a monoinfection with F. necrophorum [15]. ...
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... 27 Amplification by PCR of the lkt promoter and the hemagglutinin gene differentiated F. necrophorum subspecies from other Fusobacterium spp. 10 In the current study, the inconsistency in the band produced by F. n. subsp. funduliforme with fund primers may represent polymorphisms in the sequence of this virulence gene. ...
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